Abstract

Erythropoietin (EPO) acts on erythroid progenitor cells to promote their survival and differentiation to mature erythrocytes. Along with this canonical role, EPO is also reported to modulate energy metabolism, resulting in improved glucose tolerance and insulin sensitivity. EPO also stimulates the production of the hormone erythroferrone (ERFE) which acts to suppress hepcidin production, thus increasing dietary iron absorption and mobilizing stored iron for use in erythropoiesis. ERFE (initially termed myonectin) was also reported have an effect on systemic lipid metabolism by promoting the clearance of nonesterifed fatty acids (NEFA) from circulation. As increased levels of circulating NEFA blunt insulin sensitivity and impair glucose tolerance, ERFE-induced clearance of NEFA after EPO administration would have a beneficial effect on glucose metabolism. The aim of this study was to determine if the known metabolic effect of EPO treatment on glucose homeostasis is mediated by ERFE, produced in response to EPO. Mice lacking Erfe did not differ from wild-type mice in blood lipid parameters, blood glucose, and glucose or insulin tolerance at baseline or after chronic EPO treatment. Additionally, hepcidin suppression and the response of erythrocyte parameters to chronic EPO treatment were unaffected by the absence of Erfe. These findings suggest that the known beneficial effects of EPO on glucose metabolism are not attributable to an accompanying increase in ERFE production, and that Erfe is dispensable for normal glucose homeostasis. Furthermore, our data indicate that ERFE-independent mechanisms can suppress hepcidin in response to chronically elevated EPO levels.

KEYWORDS:

Erfe−/− mice have normal blood lipid levels during both fed and fasted conditions. Serum NEFA (A), triglyceride (B), total cholesterol (C), HDL‐C (D), LDL‐C (E), and serum glucose levels (F) in male WT and Erfe−/− knockout mice that had access to food or were fasted for 16 h prior to sacrifice. Parameters were measured at 10 weeks of age in fed mice and at 18 weeks in fasted mice (n = 8–10 per group). Group means between WT and Erfe‐/‐ mice were independently compared under either fed or fasted conditions by using the student's t‐test. Different superscripts indicate statistical significance (P <0.05). Values are presented as group means ± SEM.

Serum levels of nonesterified fatty acids are not affected by physiologic concentrations of ERFE. Bone marrow mRNA expression of Erfe (A) and serum ERFE levels (B) in WT mice after chronic treatment with either EPO or saline (NaCl) (n = 5–6 per group for each sex). (C) Serum nonesterified fatty acid (NEFA) concentrations in WT and Erfe−/− mice after chronic treatment with either EPO or saline (NaCl) (n = 5–7 per group for each sex). In panels B and C data are presented as individual values from experimental animals with a line indicating the group mean. Group means between mice of the same sex were compared by using either the student's t‐test (A and B) or one‐way ANOVA (C). Asterisks indicate a statistically significant difference between groups as determined by t‐test (*P <0.05, **P <0.01, ***P <0.001) and means without a common alphabetical superscript differ significantly as determined by one‐way ANOVA (P <0.05). Data are shown as the mean ± SEM or as individual data points.

Serum levels of nonesterified fatty acids are not affected by acute EPO treatment. Bone marrow mRNA expression of Erfe (A), serum erythroferrone levels (B), and serum nonesterified fatty acid (NEFA) concentrations in wild‐type mice 15 h after treatment with a single dose of either 200 U EPO or saline (NaCl). In panels B and C, data are presented as individual values from experimental animals with a line indicating the group mean. Group means between mice of the same sex were compared by using the student's t‐test. Asterisks indicate a statistically significant difference between groups (***P <0.001). Data are shown as the mean ± SEM or as individual data points (n = 5 per group for each sex).

Erythroferrone does not affect glucose tolerance under basal conditions or after chronic treatment with EPO. Blood glucose levels during glucose tolerance testing in male (A) and female (C) mice. Curves that are not sharing a letter are significantly different as determined by two‐way repeated measures ANOVA (P <0.05). Area under the curve (AUC) analysis of blood glucose values for male (B) and female (D) mice during glucose tolerance testing. Groups of the same sex without a common alphabetical superscript differ significantly as determined by one‐way ANOVA (P <0.05). Data are shown as the mean ± SEM (n = 6–8 per group for each sex).

Erythroferrone does not affect insulin tolerance under basal conditions or after chronic treatment with EPO. Blood glucose levels during insulin tolerance testing in male (A) and female (C) WT and Erfe‐/‐mice chronically treated with either 200 U EPO or saline (NaCl). Curves not sharing a letter are significantly different as determined by two‐way repeated measures ANOVA (P <0.05). Area under the curve (AUC) analysis of blood glucose values for male (B) and female (D) mice during insulin tolerance testing. Groups without a common alphabetical superscript differ significantly from other groups of the same sex as determined by one‐way ANOVA (P <0.05). Data are shown as the mean ± SEM (n = 6–8 per group for each sex).

Stimulation of erythropoiesis by chronic erythropoietin treatment is preserved in mice lacking erythroferrone. Hemoglobin (A), red blood cell (B), hematocrit (C), and mean corpuscular volume (D) levels measured in male and female mice chronically treated with either 200 U EPO or saline (NaCl). Groups without a common alphabetical superscript differ significantly from other groups of the same sex as determined by one‐way ANOVA (P <0.05). Data are shown as the mean ± SEM (n = 5–6 per group for each sex).

Liver iron content and hepcidin expression are not different between WT and Erfe−/− mice under basal conditions or after chronic EPO treatment. (A) Serum hepcidin concentrations in WT and Erfe−/− mice chronically treated with either EPO or saline (NaCl). Groups were analyzed by three‐way ANOVA (genotype, sex, treatment), and there was no significant interaction between factors (P <0.05). (B) Liver nonheme iron concentrations in WT and Erfe−/− mice chronically treated with either EPO or saline (NaCl). Groups without a common alphabetical superscript differ significantly from other groups of the same sex as determined by one‐way ANOVA (P <0.05). Data are shown as the mean ± SEM (n = 5–7 per group for each sex).